Influence of Neutrophils on Blood Clot Retraction

BACKGROUND: Immunothrombosis, or inflammatory thrombosis, largely determines the course and outcomes of severe infectious and autoimmune diseases. Inflammatory thrombi are rich in neutrophils; however, how inflammatory cells affect the contraction (retraction) of a blood clot, an important process that influences the course and outcomes of hemostatic disorders, remains unknown.

AIM: This study aimed to assess the effect of activated neutrophils and neutrophil extracellular traps on the rate and degree of blood clot retraction mediated by activated platelet contraction.

METHODS: Isolated human neutrophils were activated with phorbol 12-myristate 13-acetate to induce the formation of neutrophil extracellular traps, which were visualized using immunofluorescence (citrullinated histone H3 and DNA) and scanning electron microscopy. Thrombin-induced clots were formed for three sample groups from whole blood (n = 5 for each group) or platelet-rich plasma (n = 4 or n = 5 for each group): (1) without added neutrophils (control), (2) with nonactivated neutrophils, and (3) with activated neutrophils. Clot-retraction kinetics were recorded optically. The final degree of retraction was the difference between the initial and final clot size, expressed as a percentage of the initial value. Statistical analysis was performed using the Student’s t-test, one-way analysis of variance with a Sidak post hoc test, and the Friedman test. The significance level was set at 95%.

RESULTS: Phorbol 12-myristate 13-acetate induced the activation of neutrophils and formation of neutrophil extracellular traps, which merged into the fibrin structure. When added to whole blood or platelet-rich plasma, activated neutrophils significantly (p < 0.05) increased the final degree of clot retraction (50% ± 5% in blood and 86% ± 3% in platelet-rich plasma) compared with nonactivated neutrophils (45% ± 3% and 80% ± 1%, respectively). The kinetic parameters of retraction demonstrated accelerated contraction in the presence of activated neutrophils (greater mean velocity in platelet-rich plasma: 0.070%/s ± 0.003%/s vs. 0.065%/s ± 0.001%/s; p = 0.03). The stimulatory effect of activated neutrophils on clot retraction disappeared following neutrophil extracellular trap degradation with deoxyribonuclease I, in whole blood (degree of retraction 44% ± 5% vs. 54% ± 2%; p = 0.02) and platelet-rich plasma (82% ± 3% vs. 87% ± 2%; p = 0.03), indicating a key role of neutrophil extracellular traps within clots.

CONCLUSION: Activated neutrophils accelerate and enhance blood clot retraction. This effect is mediated by the formation of neutrophil extracellular traps that merge into the fibrin network.